Microbiology, 2021

7.5 • Using Biochemistry to Identify Microorganisms 279
results often inform decisions about treatment that directly affect patient outcomes. For example, cases of food
poisoning require accurate identification of the causative agent so that physicians can prescribe appropriate
treatment. Likewise, it is important to accurately identify the causative pathogen during an outbreak of disease
so that appropriate strategies can be employed to contain the epidemic.
There are many ways to detect, characterize, and identify microorganisms. Some methods rely on phenotypic
biochemical characteristics, while others use genotypic identification. The biochemical characteristics of a
bacterium provide many traits that are useful for classification and identification. Analyzing the nutritional
and metabolic capabilities of the bacterial isolate is a common approach for determining the genus and the
species of the bacterium. Some of the most important metabolic pathways that bacteria use to survive will be
discussed in Microbial Metabolism. In this section, we will discuss a few methods that use biochemical
characteristics to identify microorganisms.
Some microorganisms store certain compounds as granules within their cytoplasm, and the contents of these
granules can be used for identification purposes. For example, poly-β-hydroxybutyrate (PHB) is a carbon- and
energy-storage compound found in some nonfluorescent bacteria of the genus Pseudomonas. Different
species within this genus can be classified by the presence or the absence of PHB and fluorescent pigments.
The human pathogen P. aeruginosa and the plant pathogen P. syringae are two examples of fluorescent
Pseudomonas species that do not accumulate PHB granules.
Other systems rely on biochemical characteristics to identify microorganisms by their biochemical reactions,
such as carbon utilization and other metabolic tests. In small laboratory settings or in teaching laboratories,
those assays are carried out using a limited number of test tubes. However, more modern systems, such as the
one developed by Biolog, Inc., are based on panels of biochemical reactions performed simultaneously and
analyzed by software. Biolog’s system identifies cells based on their ability to metabolize certain biochemicals
and on their physiological properties, including pH and chemical sensitivity. It uses all major classes of
biochemicals in its analysis. Identifications can be performed manually or with the semi- or fully automated
instruments.
Another automated system identifies microorganisms by determining the specimen’s mass spectrum and then
comparing it to a database that contains known mass spectra for thousands of microorganisms. This method
is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and
uses disposable MALDI plates on which the microorganism is mixed with a specialized matrix reagent (Figure
7.26). The sample/reagent mixture is irradiated with a high-intensity pulsed ultraviolet laser, resulting in the
ejection of gaseous ions generated from the various chemical constituents of the microorganism. These
gaseous ions are collected and accelerated through the mass spectrometer, with ions traveling at a velocity
determined by their mass-to-charge ratio (m/z), thus, reaching the detector at different times. A plot of
detector signal versus m/z yields a mass spectrum for the organism that is uniquely related to its biochemical
composition. Comparison of the mass spectrum to a library of reference spectra obtained from identical
analyses of known microorganisms permits identification of the unknown microbe.
Figure 7.26 MALDI-TOF methods are now routinely used for diagnostic procedures in clinical microbiology laboratories. This technology
is able to rapidly identify some microorganisms that cannot be readily identified by more traditional methods. (credit “MALDI plate photo”:
modification of work by Chen Q, Liu T, Chen G; credit “graphs”: modification of work by Bailes J, Vidal L, Ivanov DA, Soloviev M)